Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in combination with associated sequences (cas) constitute the CRISPR-Cas system, which confers adaptive immunity in many bacteria. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and phages, as novel “spacers.”
Bacteria used in industrial settings for fermentation purposes are often times challenged by ubiquitous bacteriophage, occasionally interfering with manufacturing processes and product quality (Barrangou and Horvath. 2012. Annu. Rev. Food Sci. Technol. 3:143-162). Although phage resistance has historically relied on diversifying starter cultures and formulation based on the occurrence of phage defense systems such as restriction-modification and abortive infection (Barrangou and Horvath. 2012. Annu. Rev. Food Sci. Technol. 3:143-162), the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR) and associated sequences (cas) have shown promise for phage resistance. CRISPR-Cas systems consist of arrays of short DNA repeats interspaced by hypervariable sequences, flanked by cas genes, that provide adaptive immunity against invasive genetic elements such as phage and plasmids, through sequence-specific targeting and interference (Barrangou et al. 2007. Science. 315:1709-1712; Brouns et al. 2008. Science 321:960-4; Horvath and Barrangou. 2010. Science. 327:167-70; Marraffini and Sontheimer. 2008. Science. 322:1843-1845: Bhaya et al. 2011. Annu. Rev. Genet. 45:273-297: Terns and Terns. 2011. Curr. Opin. Microbiol. 14:321-327: Westra et al. 2012. Annu. Rev. Genet. 46:311-339; Barrangou R. 2013. RNA. 4:267-278). Typically, invasive DNA sequences are acquired as novel “spacers” (Barrangou et al. 2007. Science. 315:1709-1712), each paired with a CRISPR repeat and inserted as a novel repeat-spacer unit in the CRISPR locus. Subsequently, the repeat-spacer array is transcribed as a long pre-CRISPR RNA (pre-crRNA) (Brouns et al. 2008. Science 321:960-4), which is processed into small interfering CRISPR RNAs (crRNAs) that drive sequence-specific recognition. Specifically, crRNAs guide nucleases towards complementary targets for sequence-specific nucleic acid cleavage mediated by Cas endonucleases (Gameau et al. 2010. Nature. 468:67-71; Haurwitz et al. 2010. Science. 329:1355-1358; Sapranauskas et al. 2011. Nucleic Acid Res. 39:9275-9282; Jinek et al. 2012. Science. 337:816-821; Gasiunas et al. 2012. Proc. Natl. Acad Sci. 109:E2579-E2586; Magadan et al. 2012. PLoS One. 7:e40913: Karvelis et al. 2013. RNA Biol. 10:841-851). These widespread systems occur in nearly half of bacteria (˜46%) and the large majority of archaea (˜90%). They are classified into three main CRISPR-Cas systems types (Makarova et al. 2011. Nature Rev. Microbiol. 9:467-477; Makarova et al. 2013. Nucleic Acid Res. 41:4360-4377) based on the cas gene content, organization and variation in the biochemical processes that drive crRNA biogenesis, and Cas protein complexes that mediate target recognition and cleavage.
The pickle industry relies on the use of naturally occurring bacteria for the fermentation of cucumbers in large industrial tanks (Franco et al. 2012. Appl. Environ. Microbiol. 78:1273-1284). To control the diverse microbiota naturally associated with pickles, and preclude spoilage by undesirable microorganisms, salting and brining are implemented in industrial settings. Unfortunately, acid- and halo-tolerant lactic acid bacteria often times contaminate the pickling process, resulting in a secondary fermentation, which spoils the product by generating undesirable attributes (Id). Amongst commonly encountered bacterial contaminants, Lactobacillus buchneri has been repeatedly associated with spoilage of fermenting pickles (Franco et al. 2012. Appl. Environ. Microbiol. 78:1273-1284; Johaningsmeier et al. 2012. J. Food Sci. 77:M397-M404). Recent advances in genome sequencing in this species have shed light on the molecular underpinnings that allow L. buchneri to withstand the pickling process. In particular, determining the complete genome sequences of strains NRRL B-30929 and CD034 (Liu et al. 2011. J. Bacteriol. 193:4019-4020; Eikmeer et al. 2013. J. Bacteriol. 167:334-343; Heinl et al. 2012. J. Bacteriol. 161:153-166) has established several genetic loci for substrate utilization pathways (notably lactate and carbohydrates), including the ability to convert lactic acid into acetic acid (Heinl et al. 2012. J. Bacteriol. 161:153-166) and 1,2-propanediol (Johaningsmeier et al. 2012. J. Food Sci. 77:M397-M404). Conversely, the biochemical properties of this robust bacterium have been exploited for silage inoculation to control yeast and mold growth under anaerobic conditions during the fermentation of corn, barley, wheat, and other grains into animal fodder (Heinl et al. 2012. J. Bacteriol. 161:153-166; Dreihuis et al. 1999. J Appl Microbiol. 87:583-594: Schmidt and Kung. 2010. J. Dairy Sci. 94:1616-1624).
Accordingly, there is a need for the development of methods for typing, identifying and detecting this important organism, L. buchneri, as well as for modulating the resistance of L. buchneri to invasive organisms, such as bacteriophage.